The chief function of pancreatic acinar cells is the secretion of digestive enzymes and a small amount of isotonic fluid. Both functions are regulated by hormones and neurotransmitters that use Ca2+ as the second messenger. At the same time, aberrant Ca2+ signaling is the nodal point in all forms of pancreatitis. The hypothesis of this proposal is that specifically aberrant Ca2+ influx by store-operated Ca2+ channels (SOCs) is responsible for pancreatitis. Therefore, understanding regulation and function of Ca2+ influx channels is critical for understanding acinar cell function and dysfunction in the context of pancreatitis. Acinar cells have several Ca2+ influx channels, TRPC1, TRPC3, TRPC4, TRPC6 and Orai1, all of which are gated by the ER Ca2+ sensor STIM1. TRPCs are also gated by the scaffold Homer1 that inhibits their activity. Considering the involvement of SOCs in pancreatitis, the overall goal of this proposal is to understand gating mechanism of the pancreatic SOCs by STIM1 and Homer1 and their role in pancreatitis. This will be achieved in four aims: Aim 1: Decipher the molecular mechanism of TRPCs gating by STIM1 and Homer1. Homer1 interacts with a PPXF motif to keep the channels in a close state. STIM1 opens the TRPCs by interacting with two conserved residues that are only 4 aresidues upstream of the Homer1 binding motif. We propose to determine a) the gating mechanism of TRPCs by STIM1;b) how Homer1 and STIM1 function in tandem to gate the TRPCs. Aim 2: Determine molecular mechanism of Orai1 gating by STIM1. Agonist-stimulated pancreatic Ca2+ influx is mediated by a combination of TRPCs and Orai1 that are differentially gated by STIM1. We discovered that the STIM1(234-535) fragment is sufficient for full activation of Orai1. Hence we will: a) determine the minimal STIM1 domain that gates Orai1;b) how STIM1 opens Orai1;c) what is the relationship between the NATIVE TRPCs and NATIVE Orai1. Aim 3: The role of TRPCs, Orai1 and STIM1 in physiological and pathological acinar cells Ca2+ signaling and exocytosis will be determined by characterizing Ca2+ signaling and exocytosis in acinar cells from TRPC1-/-, TRPC3-/-, TRPC6-/-, Orai1-/- and STIM1-/- mice. Aim 4: The role of TRPC channels, Orai1, STIM1 will be studies in vitro-induced pancreatitis and measure cellular reporters of cell stress and cell damage. Then the KO mice will be used to evaluate the role of the Ca2+ influx channels in careluin and bile acid models of pancreatitis. PUBLIC HEALTH RELEVANCE: The pancreas secretes digestive enzymes and fluid in response to stimulation by hormones and neurotransmitters. The stimulants tell the pancreatic cells how to do so by changing the concentration of Ca2+ ions within the cells. Most of the Ca2+ enters the cells from the extracellular environment. When this process is aberrant, the digestive enzymes remain trapped within the cells and the cells dye and the patient develop the disease called pancreatitis. The aims of this proposal are to understand how Ca2+ ions enter the cells and how the aberrant Ca2+ entry causes the pancreatitis. This will be achieved by studying Ca2+ entry into the cells in model cell systems and in the cells of the pancreas of mice from which the genes that are responsible for the entry of Ca2+ into the cells have been deleted.